Composition for detecting analyte

Abstract

The present invention relates to a method for detecting or measuring an analyte, in particular a protein or a peptide. The present invention provides a means capable of detecting, with high accuracy, an extremely small amount of a protein or peptide present in a target sample, and can thus be effectively used for the detection of multiple peptides and the early diagnosis of cancer therethrough.

Description

Composition for detection of analyte

[0001] The present invention relates to a detection composition having an effect of amplifying the amount of an analyte for accurate detection of an analyte such as a protein, a detection kit, and a method for detecting an analyte using the same.

[0002]

[0003] Cancer diagnosis is currently transitioning from a single-marker approach to a multi-marker approach. Diagnosing cancer by measuring the presence and quantity of markers, such as proteins or peptides, in biological samples collected from the subject offers convenience and facilitates early diagnosis. However, ensuring diagnostic accuracy requires the implementation of methods capable of accurately detecting these markers.

[0004] In particular, cancer diagnosis requires the detection of multiple markers due to its strong heterogeneity. However, when using the existing immunodiagnostic method, ELISA, to detect these multiple markers, multiple antibodies specific to each marker must be identified and positioned on a plate in separate sections. This requires that the cost increase exponentially as the number of markers increases, and the analysis procedure becomes very complex. Furthermore, due to the low efficiency of these conventional immunodiagnostic methods, a large volume of samples (such as blood) must be collected from the subject to ensure clinically acceptable diagnostic accuracy. This presents many limitations for using these existing immunodiagnostic methods for multi-marker diagnosis. Therefore, the integration of technologies such as MS / MS, which enable high-sensitivity sensing and simultaneous multiplex quantification in samples (such as blood), is essential for multi-marker sensing and quantification. However, in order to detect multiple markers for cancer diagnosis from a small amount of sample, the detection accuracy must be very high. However, unlike genetic markers composed of nucleic acids, there is no method for amplifying the detection target for protein or peptide markers. Therefore, the inventors of the present invention developed a method for amplifying protein or peptide markers, and through this, they aimed to invent a method for accurately diagnosing cancer at an early stage using only a small amount of sample.

[0005] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0006]

[0007] The method of diagnosing cancer by collecting a small sample, such as blood, from a diagnosis subject and detecting protein or peptide markers contained in the sample is very effective for early diagnosis due to its convenience. However, unlike genetic markers, there is no amplification method for peptides. Therefore, the present inventors have made extensive research efforts to improve the detection accuracy of peptide markers. As a result, when an artificial amino acid sequence that does not exist in nature is used as an amplification sequence, and this is specifically bound to the peptide marker to be detected, and the bound amplification sequence is detected instead of the peptide marker to be detected, an amplification effect can also be observed for the peptide marker. In addition, by developing an amplification sequence of a specific sequence that is particularly effective in the detection method, the inventors have discovered that cancer can be diagnosed early and with high accuracy by collecting only a small amount of sample from a diagnosis subject, thereby completing the present invention.

[0008] Accordingly, the purpose of the present invention is to provide a composition for detecting or measuring an analyte.

[0009] Another object of the present invention is to provide a kit for detecting or measuring an analyte.

[0010] Another object of the present invention is to provide a method for analyzing an analyte.

[0011] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0012]

[0013] According to one aspect of the present invention, the present invention provides a composition for detecting or measuring an analyte, comprising a binding portion that specifically binds to the analyte; and an amplification portion.

[0014] The method of diagnosing cancer by collecting a small sample, such as blood, from a diagnosis subject and detecting protein or peptide markers contained in the sample is very effective for early diagnosis due to its convenience. However, unlike genetic markers, there is no amplification method for peptides. Therefore, the present inventors have made extensive research efforts to improve the detection accuracy of peptide markers. As a result, when an artificial amino acid sequence that does not exist in nature is used as an amplification sequence, and this is specifically bound to the peptide marker to be detected, and the bound amplification sequence is detected instead of the peptide marker to be detected, an amplification effect can also be observed for the peptide marker. In addition, by developing an amplification sequence of a specific sequence that is particularly effective in the detection method, the inventors have discovered that cancer can be diagnosed early and with high accuracy by collecting only a small amount of sample from a diagnosis subject, thereby completing the present invention.

[0015] The term “analyte” used herein refers to a substance to be detected or measured through the composition of the present invention. The “analyte” may specifically be a substance present in a biological sample (or biological specimen) collected from a cancer diagnosis subject, and more specifically may include at least one selected from the group consisting of proteins, lipoproteins, glycoproteins, DNA, and RNA. However, any molecule within a living body that contains organic substances such as amino acids, nucleotides, monosaccharides, or lipids as monomers may be included without limitation.

[0016] In this specification, the term “binding portion” refers to one of the structures included in the composition for detecting or measuring an analyte of the present invention, and refers to a portion that directly binds to the analyte or indirectly binds to the analyte. The meaning of “indirectly binding to the analyte” above refers to a binding structure having a connection state in which a separate additional configuration exists between the binding portion and the analyte, and specifically refers to a binding structure in which an antibody that specifically binds to the analyte directly binds to the analyte through the antigen binding portion, and a connection state in which the binding portion is connected to a portion other than the antigen binding portion of the antibody.

[0017] In this specification, the term “amplification unit” refers to a polymer formed by repeating and connecting specific units multiple times, which is one of the structures included in the composition for detecting or measuring analytes of the present invention. The amplification unit is specifically connected to the analyte through a binding unit, and then, when detecting the analyte, a specific means (specifically, a protease, etc.) is administered to decompose it into multiple units again, so that multiple units correspond to one analyte, and consequently, multiple units are detected instead of the analyte, thereby having the effect of detecting the analyte, i.e., the effect of “amplifying” a trace amount of the analyte.

[0018] In this specification, the term “unit” may be used interchangeably with “monomer” and refers to a compound that serves as a unit used to synthesize the amplification unit. In the present invention, the unit may specifically be an amino acid, an amino acid analog, a peptide, a peptide analog, a monosaccharide, an oligosaccharide, or a polysaccharide, but is not limited thereto, and any material that can be linked to each other to form an amplification unit (polymer) and can be decomposed or separated into units when necessary may be used. The units constituting the amplification unit may be identical or different from each other.

[0019] The term “amino acid” as used herein refers to a substance that has a structure in which a basic amino group (-NH2), an acidic carboxyl group (-COOH), and a side chain (-R group) are bonded to a central carbon, which is an alpha carbon, and is capable of forming a peptide bond, and any substance having the above characteristics may be included without limitation. Therefore, the amino acid includes both those derived from living organisms and those artificially synthesized, and its constituent elements are not limited to carbon, hydrogen, oxygen, nitrogen, or sulfur, but additionally include other elements, and all forms of isomers are also included. The amino acids include not only the 20 kinds of amino acids encoded by the genes of eukaryotes and prokaryotes, but also all kinds of amino acids, more than 500 of which occur in nature.

[0020] The term “amino acid analog” in this specification refers to a substance that can be used to crosslink peptides or protein complexes instead of amino acids by forming a peptide bond, and includes all substances having an amino group (-NH2) and a carboxyl group (-COOH) in the molecule.

[0021] Specifically, the structure included in the composition for detecting or measuring the analyte of the present invention can be represented by the following formula.

[0022] [Formula 1]

[0023] [M] n -L1-N1

[0024] In the above formula, M represents a unit, and [M] n refers to an amplifier section in which the unit is repeated as many times as the integer n, and L1 is the above [M] n and N1, wherein L1 includes a direct bond or a linker connecting them, and N1 means a bonding portion.

[0025] The term “linker” as used herein refers to a compound that cross-links another compound, which may be by a chemical bond such as a covalent bond or a physical bond such as an ionic bond. A protecting group may be introduced during the cross-linking process.

[0026]

[0027] According to a specific embodiment of the present invention, the amplification unit comprises a peptide consisting of one or more amplification sequences selected from the group consisting of SEQ ID NOs: 1 to 4.

[0028] In this specification, the term “amplification sequence” means a plurality of units constituting an amplification unit (complex).

[0029]

[0030] According to a specific embodiment of the present invention, the amplification sequence comprises a peptide consisting of a sequence of SEQ ID NO: 1 to 4 as a unit, and specifically, the unit is 10 to 100000, more specifically 10 to 10000, even more specifically 10 to 5000, even more specifically 10 to 1000, even more specifically 10 to 500, even more specifically 10 to 200, more specifically 10 to 180, even more specifically 10 to 160, even more specifically 10 to 140, even more specifically 10 to 120, even more specifically 10 to 100, even more specifically 10 to 90, even more specifically 15 to 85, even more specifically 20 to 80, even more specifically 25 to 75, even more specifically 30 to A peptide that is repeated 70 times, more specifically 35 to 65 times, more specifically 40 to 60 times, more specifically 45 to 55 times, and most specifically 50 times.

[0031] In this specification, the term “unit” refers to a substance that constitutes a unit of a complex, and specifically refers to a unit substance peptide that constitutes an amplification portion of the complex.

[0032]

[0033] According to a specific embodiment of the present invention, the repeating amplification sequence is decomposed into a plurality of the above units by a protease.

[0034] In this specification, the term “protease” may be used interchangeably with “peptidase”, and refers to an enzyme that hydrolyzes peptide bonds between peptides that constitute a protein. The protease that acts on the N-terminus or C-terminus of the peptide chain that constitutes the protein to release amino acids in the bonded order is called an exopeptidase, and the enzyme that acts on the peptide bond within the peptide chain is called an endopeptidase. The peptide hydrolase can be used to specifically hydrolyze only the peptide bond of a specific amino acid.

[0035] In the present invention, the protein decomposing enzyme may be at least one selected from the group consisting of trypsin, chymotrypsin, thrombin, plasmin, subutirilsin, thermolysin, pepsin, and glutamyl endopeptidase, and specifically, may be at least one selected from the group consisting of trypsin, chymotrypsin, subutirilsin, thermolysin, and glutamyl endopeptidase, but is not limited thereto.

[0036] In the present invention, the process of decomposing or separating the complex “amplifier” into its units may be performed catalytically or pH- or temperature-specifically using a decomposing enzyme including the aforementioned protein decomposing enzyme, or may be performed without being restricted by pH or temperature using a synthetic catalyst.

[0037] In the present invention, the synthetic catalyst may be, but is not limited to, an artificial metal enzyme, an organoenzyme, or a reducing agent that cleaves a disulfide bond.

[0038] In the present invention, the artificial metalloproteases include, but are not limited to, water-soluble catalysts using copper (II), cobalt (III), iron (III), palladium (II), cerium (IV), etc. as the center of the catalyst, or those in which a copper (II) complex is attached to a support.

[0039] In the present invention, the organic artificial proteases may be those that attach a functional group to a silica support or a polystyrene support, but are not limited thereto.

[0040]

[0041] According to a specific embodiment of the present invention, the amplification unit additionally includes a peptide for quality control (QC).

[0042] The term “quality control (QC) peptide” as used herein refers to a peptide that can be included in an amplification unit, and has a different sequence from the monomer constituting the amplification unit, and a sequence that does not match the human protein amino acid sequence. The “quality control (QC) peptide” is simultaneously separated when the amplification unit is decomposed or separated into the monomers constituting it, and is used to perform quality control (QC). The detection of the quality control (QC) peptide is used to confirm whether the F50 protein has been normally decomposed into monomers, and is also used to compare the amplification ability by the amplification sequence. Here, comparing the amplification ability means that when the amplification sequence constituting the amplification unit is decomposed by an enzyme such as trypsin or other cleavage means, the quality control peptide is also decomposed together, and quality control can be performed by comparing the peak intensity of the amplification sequence based on the peak intensity of the quality control peptide during mass spectrometry. More specifically, since the peptide for quality control of the present invention is repeated once, and the F50 sequence for amplification of the present invention is repeated 50 times, when the intensity of the mass analysis peak by the peptide for quality control is set to 1, it is confirmed that the intensity of the mass analysis peak by F50 is detected as (approximately) 50, thereby confirming that the separation of the sequences for amplification and the amplification through the same have been performed appropriately, and through this, a quality control operation can be performed.

[0043] Specifically, the peptide for quality control (QC) may be a peptide having a sequence different from a peptide represented by a sequence of SEQ ID NO: 1 to 4 and a peptide represented by a human protein amino acid sequence, and more specifically, may be a peptide represented by SEQ ID NO: 5.

[0044] As used herein, the term “a sequence different from a peptide represented by a human protein amino acid sequence” refers to a peptide having a sequence that is different from a protein or peptide sequence naturally observed in an actual human, and thus does not cause an “interference phenomenon” in which the detection accuracy is lowered due to confusion with a naturally existing protein or peptide when detecting an analyte. Therefore, “a sequence different from a peptide represented by a human protein amino acid sequence” refers to an artificial sequence that does not have 90% or more homology with a naturally existing protein or peptide, more specifically, refers to an artificial sequence that does not have 80% or more homology, and most specifically, refers to an artificial sequence that does not have 70% or more homology.

[0045]

[0046] According to a specific embodiment of the present invention, the binding portion includes a binding region that specifically binds to an analyte; or an antibody that specifically binds to the analyte.

[0047] In the present invention, the binding unit can directly and specifically bind to the analyte to be detected, and can also form a bond by specifically binding to an antibody that specifically binds to the analyte. Since the binding unit specifically binds to the analyte through direct binding or indirect binding via an antibody, the analyte and the amplification unit correspond one-to-one, and thus, instead of detecting a trace amount of the analyte, the analyte can be detected using the amplification unit that corresponds one-to-one. Therefore, when using the detection or measurement composition of the present invention, an analyte such as a peptide can also enjoy an effect equivalent to amplifying the amount of a sample.

[0048] In the present invention, the means for specifically binding the binding portion and the analyte directly or indirectly may use an antibody, but is not limited thereto and any means for specifically binding may be used.

[0049]

[0050] According to a specific embodiment of the present invention, the binding portion additionally includes a region for protein purification.

[0051] As used herein, the term "region for protein purification" refers to a region that can be included in the binding portion, and refers to a portion used in a purification process to isolate only the composition for detecting or measuring the analyte of the present invention that has bound to the analyte. Specifically, the protein purification of the present invention may utilize a method such as using an affinity tag for the protein to be purified, but is not limited thereto and any method used for protein purification may be utilized.

[0052]

[0053] According to a specific embodiment of the present invention, the region for protein purification is an affinity tag.

[0054] As used herein, the term "affinity tag" refers to a tag material used to isolate or purify a target substance by utilizing the specific interaction between a protein and a specific ligand. Specifically, in the present invention, the affinity tag may be one or more selected from the group consisting of a His tag, a Myc tag, an HA tag, a GST tag, and a FLAG tag.

[0055]

[0056] According to a specific embodiment of the present invention, the His tag comprises 1 to 20 histidines, specifically 2 to 19, more specifically 3 to 18, more specifically 4 to 17, more specifically 5 to 16, more specifically 6 to 15, more specifically 6 to 14, more specifically 6 to 13, and most specifically 6 to 12.

[0057]

[0058] According to a specific embodiment of the present invention, the binding portion additionally includes a region capable of biotinylation.

[0059]

[0060] According to a specific embodiment of the present invention, the biotinylated region is an amino acid containing an amine group.

[0061]

[0062] According to a specific embodiment of the present invention, the amino acid containing the amine group is lysine.

[0063]

[0064] According to a specific embodiment of the present invention, the biotinylation region may include at least one amino acid containing an amine group. Specifically, the biotinylation region includes 1 to 50 amino acids containing an amine group, specifically 5 to 45 amino acids, more specifically 10 to 40 amino acids, even more specifically 10 to 30 amino acids, even more specifically 15 to 35 amino acids, even more specifically 15 to 30 amino acids, even more specifically 16 to 25 amino acids, even more specifically 17 to 23 amino acids, even more specifically 18 to 21 amino acids, even more specifically 18 to 20 amino acids, and most specifically 19 amino acids.

[0065]

[0066] According to another aspect of the present invention, the present invention provides a kit for detecting or measuring an analyte comprising the composition for detecting or measuring of the present invention.

[0067]

[0068] According to a specific embodiment of the present invention, the kit is for mass spectrometry.

[0069] In this specification, the term “mass spectrometry” means an analytical technique that can selectively separate, detect, and quantify a specific analyte in a biological sample and monitor changes in its concentration through mass-spectrometry (MS) using a specific mass-to-charge ratio (m / z) of the substance.

[0070]

[0071] According to another aspect of the present invention, the present invention comprises the steps of reacting the above-described composition with an analyte;

[0072] A step of separating or purifying a combination of the above-described composition and the analyte from the product after the above reaction;

[0073] A step of treating the above complex with a protease; and

[0074] A method for analyzing an analyte is provided, including a step of detecting an amplification sequence generated by decomposition by the above protease.

[0075] Since the meanings of the terms “analyte,” “protein decomposition enzyme,” and “amplification sequence” in this specification have already been described above, their description is omitted to avoid excessive duplication.

[0076]

[0077] According to a specific embodiment of the present invention, the step of separating or purifying the conjugate may be performed by enzyme-linked immunosorbent assay (ELISA), but is not limited thereto, and any method known in the technical field to which the present invention pertains for separating or purifying only the target substance may be used.

[0078] The term “enzyme-linked immunosorbent assay (ELISA)” as used herein refers to a method of quantitatively measuring an antigen (or antibody) by confirming the strength of an antigen-antibody reaction using the enzyme activity of an enzyme-labeled antibody (or antigen) as an indicator. Specifically, the enzyme-linked immunosorbent assay is a direct ELISA, an indirect ELISA, a sandwich ELISA, or a competitive ELISA.

[0079] In this specification, the term “direct enzyme-linked immunosorbent assay” refers to an enzyme-linked immunosorbent assay that uses a detection antibody to which a signal molecule, such as an antibody or fluorescent substance, is directly bound (labeled), as a type of enzyme-linked immunosorbent assay.

[0080] In this specification, the term “indirect enzyme-linked immunosorbent assay” refers to an enzyme-linked immunosorbent assay that uses a secondary antibody containing a signal molecule that specifically binds to the primary antibody together with the primary antibody, instead of using a primary antibody that binds to an antigen.

[0081] In this specification, the term “sandwich enzyme-linked immunosorbent assay” refers to an enzyme-linked immunosorbent assay performed by first fixing a detection target (antigen, etc.) to the bottom of a plate, administering the antigen to perform an antigen-antibody reaction with the antibody fixed to the bottom, and then administering a detection antibody.

[0082] In this specification, the term “competitive enzyme-linked immunosorbent assay” refers to an enzyme-linked immunosorbent assay performed by competitively reacting different antigens having the same antibody binding site with an antibody.

[0083]

[0084] According to a specific embodiment of the present invention, the protease is trypsin.

[0085]

[0086] According to a specific embodiment of the present invention, the detection of the amplification sequence is performed by one or more methods selected from the group consisting of protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, aukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), western blotting, and multiple reaction monitoring (MRM).

[0087]

[0088] The features and advantages of the present invention are summarized as follows:

[0089] (a) The present invention provides a method for detecting or measuring an analyte, particularly a protein or peptide.

[0090] (b) The present invention provides a means for detecting proteins or peptides present in trace amounts in a target sample with high accuracy, and thus can be usefully used for the detection of multiple peptides and the early diagnosis of cancer through the same.

[0091]

[0092] Figure 1 is a diagram illustrating the structure and sequence of the recombinant protein F50 of the present invention. Figure 1a is a diagram depicting the protein tertiary structure predicted using the Alphafold program for the structure of the recombinant protein F50 of the present invention. Figure 1b is a diagram exemplifying the amino acid sequence that the F50 protein may have.

[0093] Figure 2 illustrates the experimental results confirming the characteristics of recombinant protein F50 during the production process. Figure 2a illustrates the results of confirming the protein size of biotinylated F50 (ESF50 equivalent) in a PAGE gel. Figure 2b illustrates the results of an experiment confirming the biotinylation of F50 using an Octet SA biosensor.

[0094] Figure 3 is a drawing showing the results of an experiment in which digested F50 was injected into LC-MS / MS and the amplification sequence FTPVR was detected. The results of calculating the ratio of the FTPVR heavy peptide standard measurement value and the serially diluted F50 measurement value are also shown.

[0095] Figure 4 illustrates the results of LC-MS / MS analysis using F50 applied to the CA19-9 ELISA system. It was confirmed that the amount of FTPVR detected increased linearly as the CA19-9 standard concentration increased, confirming the amplification effect of the target peptide detected by F50.

[0096] Figure 5 is a graph showing the results of LC-MS / MS analysis of F50 and human serum together after desalting after CA19-9 ELISA. Figure 5a shows the results when desalted F50 was used without mixing with serum, and Figure 5b shows the results when desalted F50 was used after mixing with serum.

[0097]

[0098] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0099]

[0100] Example

[0101]

[0102] Selection of AMPI-tag repeat sequences and production of various tags

[0103] Purpose of development

[0104] When analyzing blood samples using MS / MS, some proteins are difficult to detect due to their complexity and low concentrations of target proteins. The purpose of developing AMPI-tags is to amplify the signals of these proteins in MS / MS. The present invention was developed by biosynthesizing a tag with a highly repetitive amino acid sequence that does not exist in nature and incorporating it into an existing ELISA system. This amplifies the signal of target proteins in MS / MS, thereby improving the detection efficiency and accuracy of ultra-trace proteins.

[0105] Specifically, the purpose of the present invention is to develop a specific repetitive tag sequence among AMPI tags that can be utilized for practical diagnosis. In other words, through the present invention, the purpose is to develop a sequence that does not cause interference in human blood, does not overlap, and exhibits high efficiency in mass spectrometry, and to establish a manufacturing method and testing protocol for such sequences.

[0106]

[0107] Selection of AMPI-tag sequence

[0108] The human genome sequence was obtained from the Uniprot database, and all possible combinations of pentamer sequences were extracted from it. By comparing this with the list of all possible pentamers for each amino acid combination, a list of orthogonal pentamers not found in humans was obtained. Of these, only sequences that ended in R or K as the last amino acid and were digestible by trypsin were retained. Of the remaining approximately 100 pentamers, FTPVR, SLVPR, GSVFR, and GYEFR were ultimately selected after considering hydrophobicity and interference with human serum.

[0109]

[0110] Development of recombinant proteins with repetitive sequences

[0111] (1) Strain production

[0112] In order to produce an artificial sequence in which the selected sequences (FTPVR, SLVPR, GSVFR, GYEFR) are linked in a high-repetition manner in Escherichia coli, the FTPVR repeat sequence was produced alone and also in the form of a fusion with various proteins. After producing a 50-repetition FTPVR sequence by synthesis, it was amplified using primers and inserted into the pQE80L vector using the Infusion HD cloning kit. The produced vector was transformed into the E. coli DH5a strain, and the cloning was verified through colony PCR and sequencing.

[0113] The unit sequences constituting the amplification sequence of the present invention are as follows:

[0114] (1) FTPVR (SEQ ID NO: 1)

[0115] (2) SLVPR (SEQ ID NO: 2)

[0116] (3) GSVFR (SEQ ID NO: 3)

[0117] (4) GYEFR (SEQ ID NO: 4)

[0118]

[0119] (2) Manufacturing of joints

[0120] 1) The binding site where the AMPI-tag binds to the analyte or target antibody was designed to have a structure that facilitates biotinylation, and was constructed to contain at least two lysines whose functional group is in the amine form.

[0121] 2) In addition, for purification after strain expression, the strain is produced by sequentially expressing six or more histidines that specifically bind to nickel beads.

[0122]

[0123] (3) Manufacturing of amplifier

[0124] 1. The ends of the repetitive sequences that are to be cleaved into the measurement material are arranged with amino acids that can be cleaved by a protease such as trypsin.

[0125] 2. The repeat sequence forms an arrangement of three or more amino acids.

[0126] 3. For quality control (QC), the sequence is composed of three or more amino acids that are not identical to the human protein amino acid sequence and are sequences of other sequences that are cleaved together in addition to the repetitive sequence.

[0127] An example of a sequence that can be used as a quality control (QC) sequence of the present invention is as follows:

[0128] (1) SDVDLQPK (SEQ ID NO: 5)

[0129]

[0130] (4) Production results

[0131] As a result of verification through E. coliDH5a, a recombinant protein with 50 FTPVR repeat sequences was developed in a soluble form and named F50 (or ESF50) (Fig. 1). In addition, a protein F100 with 100 FTPVR repeat sequences, a recombinant protein with 50 SLVPR repeat sequences, a recombinant protein with 20 GSVFR repeat sequences, and a recombinant protein with 20 GYEFR repeat sequences were developed.

[0132] The specific sequences of the binding portion, linker, and amplification portion produced through the present invention may be as follows.

[0133]

[0134] a) Joint:

[0135] MRGSHHHHHHGSACDIVLTQTPSSLPVSVGEKVTMTCKSSQTLLYSNNQKNYLAWYQQKPGQSPKLLISWAFTRKSGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQQYSNYPWTFGGGTRLEIKRGGGGSG GGGSGGGGSGGGGSEVQLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNSGKSPEWIGHISSSYATSTYNQKFKNKAAFTVDTSSSTAFMQLNSLTSEDSAVYYCVRSGNYEEYAMDYWGQGTSVTVSS (SEQ ID NO: 6)

[0136] The above “HHHHHH” is a His tag (SEQ ID NO: 9).

[0137]

[0138] b) Linker:

[0139] GGGGSASQQDSD (SEQ ID NO: 7)

[0140]

[0141] c) Amplifier:

[0142] FTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFT PVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRFTPVRITCKSDVDLQPKLN (SEQ ID NO: 8)

[0143] The above “SDVDLQPK” is the amino acid sequence (SEQ ID NO: 5) of a portion constituting the peptide for quality control (QC) of the present invention.

[0144]

[0145] (5) Protein production

[0146] E. coli DH5a strain containing the F50 production vector was mass-cultured, cells were disrupted, and the F50 protein was extracted from the soluble fraction using a His-tag. The extracted protein was concentrated using an Amicon filter 50K, and F50 was biotinylated according to the biotinylation kit manual. Afterwards, the remaining biotin was removed using an Amicon filter 50K, and the biotinylated F50 was secured.

[0147]

[0148] F50 performance check

[0149] (1) ELISA-MS

[0150] The experiment was conducted according to the CA19-9 ELISA kit manual up to and including the biotinylated CA19-9 antibody treatment, but the CA19-9 antigen from Abbexa was used.

[0151]

[0152] (2) Sample pretreatment

[0153] In the well, denaturation was performed with urea, reduction was performed to break disulfide bonds with DTT, and alkylation was performed with IAA. Afterwards, digestion was performed with trypsin, and after desalting, analysis was performed by LC-MS / MS.

[0154]

[0155] Reagent Information

[0156] The heavy peptide standards used in this study were synthesized at the Good Manufacturing Practice (GMP) facility of Bertis Co., Ltd. (Republic of Korea). Stock solutions of each heavy peptide standard were prepared at a concentration of 1 mg / mL using ultrapure water, stored in a deep freezer at -80°C, and diluted with ultrapure water as needed. Other reagent information is listed in Table 1.

[0157]

[0158] Standard product manufacturer FTPVR{Arg(13C6,15N4)} 95.1% Bertis, Korea Reagent manufacturer Dithiothreitol (DTT) Thermo Fisher, USAIodoacetamide (IAA)Sigma-Aldrich, USAAmmonium bicarbonate (ABC)Sigma-Aldrich, USAUreaGlentham, UKTrypsinPromega, USATrifluoroacetic acid (TFA)Thermo Fisher, USAFormic acidThermo Fisher, USAAcetonitrile (ACN)Sigma-Aldrich, USACHAPSGlentham, UKBiotinylation kitAbcam, UKCA19-9 antigenProspec Bio, IsraelCA19-9 ELISA kitRayBiotech, USAStreptavidinBertis, KoreaWater (HPLC grade)Fisher scientific, USAAmicon filter (50K)Millipore, USAC18 column (0.5 x 150 mm, 3.5 um, 300 Å)Agilent, USA

[0159]

[0160] Mass spectrometry conditions

[0161] The LC-MS / MS (Liquid chromatography-tandem mass spectrometry) system used was a Qtrap 5500 from Sciex (USA), and positive mode MRM (Multiple Reaction Monitoring) analysis was performed using the Analyst 1.7.2 program. Transition information is shown in Table 2.

[0162]

[0163] MRM transitionsProteinSequenceQ3 ionAnalyte / ISQ1(m / z)Q3(m / z)DPCEF50FTPVR+2y3Analyte310.182371.24035.6415.0IS315.186381.24835.6415.0

[0164]

[0165] F50 production and performance verification results

[0166] F50 production

[0167] As a result of loading the biosynthesized F50 onto a PAGE gel, a major band was confirmed to appear around 61.3 kDa, which is the size of the F50 protein, and almost no bands of other sizes were visible, confirming that the purity was very high (Fig. 2a). To confirm whether biotin worked well in the biotinylated F50, an Octet SA biosensor with streptavidin was used, and as a result, it was confirmed that the streptavidin of the biosensor and the biotin of F50 bound well (Fig. 2b).

[0168]

[0169] F50 performance check

[0170] Confirmation of FTPVR detection by LC-MS / MS

[0171] To determine whether FTPVR could be detected when digested F50 protein was injected into LC-MS / MS, serial dilutions of F50 were tested. A known concentration of FTPVR heavy peptide standard was injected concurrently, and the ratio was calculated. The results showed that the ratio increased linearly with the amount of F50 tested (Fig. 3).

[0172]

[0173] Analytical performance of F50 in an ELISA system

[0174] We tested a system that measures CA19-9 levels using LC-MS / MS measurements instead of the absorbance of the existing ELISA system by performing ELISA on CA19-9, a pancreatic cancer marker, and applying F50. CA19-9 standards were tested at various concentrations, and the ratio of the FTPVR values ​​detected by LC-MS / MS and the FTPVR peptide standards substituted with stable isotopes at a certain concentration was calculated as the F50 measurement value. As a result, it was confirmed that the ratio increased linearly according to the concentration of the CA19-9 antigen standard used, and the coefficient of determination (R) in the range of 0-256 U / ml 2 ) showed a very high linearity of 0.9986 (Fig. 4). Each point was measured five times and expressed as the average value.

[0175] To verify the accuracy, the theoretical value of CA19-9 was calculated from the measured value of F50 using the formula of the trend line of the graph shown in Fig. 4, and the recovery rate, which is the value obtained by subtracting the calculated value from the theoretical value and dividing it by the theoretical value, is as shown in Table 3. As a result, very high accuracy was shown in all eight concentration intervals.

[0176]

[0177] CA19-9 concentration (U / ml) F50 measured value (A) F50 theoretical value (B) recovery rate (BA) / B 0 1.43 1.42 - 0.6% 8 1.46 1.49 2.1% 12 1.58 1.53 - 3.4% 24 1.64 1.63 - 0.4% 48 1.80 1.85 2.5% 12 8 2.55 2.56 0.2% 19 23.15 3.12 - 0.9% 25 6 3.68 3.69 0.3%

[0178]

[0179] Confirmation of the matrix effect of human serum

[0180] An experiment was conducted to determine whether F50 affects the detection of FTPVR when injected into LC-MS / MS together with human serum. ELISA was performed using CA19-9 standard 128 U / ml, followed by sample pretreatment with F50, and then desalting was performed by mixing with separately pretreated human serum. This was injected into LC-MS / MS for analysis, and the serum matrix effect (signal intensity in serum (Fig. 5a) / signal intensity in solvent (Fig. 5b)) was not significant at around 81% (Fig. 5).

[0181] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for detecting or measuring an analyte, comprising a binding portion that specifically binds to the analyte; and an amplification portion.

2. In paragraph 1, A composition characterized in that the amplification unit comprises a peptide consisting of one or more amplification sequences selected from the group consisting of sequence numbers 1 to 4.

3. In paragraph 2, A composition characterized in that the amplification sequence is a peptide consisting of a sequence of sequence numbers 1 to 4 as a unit, and the unit is a peptide repeated 10 to 200 times.

4. In paragraph 3, A composition characterized in that the above-mentioned repeating amplification sequence is decomposed into a plurality of the above-mentioned units by a protease.

5. In paragraph 4, A composition characterized in that the above protein-decomposing enzyme is trypsin.

6. In paragraph 2, A composition characterized in that the amplification unit additionally includes a peptide for quality control (QC).

7. In paragraph 6, A composition characterized in that the peptide for quality control (QC) is a peptide represented by a sequence of SEQ ID NO: 1 to 4 and a peptide having a sequence different from a peptide represented by a human protein amino acid sequence.

8. In paragraph 6, A composition characterized in that the peptide for the above quality control (QC) is a peptide represented by sequence number 5.

9. In paragraph 1, A composition characterized in that the binding portion comprises a binding region that specifically binds to the analyte; or an antibody that specifically binds to the analyte.

10. In paragraph 9, A composition characterized in that the above binding portion additionally includes a region for protein purification.

11. In paragraph 10, A composition characterized in that the region for protein purification comprises an affinity tag.

12. In paragraph 11, A composition characterized in that the affinity tag is at least one selected from the group consisting of a His tag, a Myc tag, an HA tag, a GST tag, and a FLAG tag.

13. In paragraph 12, A composition characterized in that the His tag contains 6 to 12 histidines.

14. In paragraph 9, A composition characterized in that the above binding portion additionally includes a region capable of biotinylation.

15. In paragraph 14, A composition characterized in that the biotinylation-capable region is an amino acid containing an amine group.

16. In paragraph 15, A composition characterized in that the amino acid containing the above amine group is lysine.

17. In paragraph 15, A composition characterized in that the biotinylated region comprises 10 to 30 amino acids containing an amine group.

18. A kit for detecting or measuring an analyte comprising a composition of any one of claims 1 to 17.

19. In paragraph 18, A kit characterized in that the above kit is for mass spectrometry.

20. A step of reacting the composition of any one of claims 1 to 17 with the analyte; A step of separating or purifying a combination of a composition of any one of claims 1 to 17 and an analyte from a product after the above reaction; A step of treating the above complex with a protease; and A method for analyzing an analyte, comprising a step of detecting an amplification sequence generated by decomposition by the above protease.

21. In paragraph 20, A method characterized in that the step of separating or purifying the above complex is performed by enzyme-linked immunosorbent assay (ELISA).

22. In paragraph 21, A method characterized in that the above enzyme-linked immunosorbent assay is a direct enzyme-linked immunosorbent assay (ELISA), an indirect enzyme-linked immunosorbent assay (Indirect ELISA), a sandwich enzyme-linked immunosorbent assay (Sandwich ELISA), or a competitive enzyme-linked immunosorbent assay (Competitive ELISA).

23. In paragraph 20, A method characterized in that the above protease is trypsin.

24. In paragraph 20, A method characterized in that the detection of the above amplification sequence is performed by at least one method selected from the group consisting of protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, aukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), western blotting, and multiple reaction monitoring (MRM).

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